srjcbiologybeckonsyoufandomcom-20200213-history
M5 Transformation Lab
***SPRING 2018***** Had many LB leftover, talk to Steven about Expected #s = Here is a LINK to the Bio Rad pGLO transformation full lab manual Here is a LINK to the STUDENT MANUAL for Micro 5 Transformation Lab Lab 1 Procedures LB PLATES - 3 days before Prepare media and pour LB Plates # Figure out how much LB media you will need by adding up all of the plates # Heat up and aliquot the 3 types into 3 different flasks (need to make both LB+amp and LB+a+a type medias in exact amounts so you can properly add the amounts of ampicillin and arabinose to those flasks after cooling) # Autoclave then pour the LB plates while cooling the other 2 media flasks to 50 degrees Celcius # After cooling, add ampicillin to both flasks and arabinose to the LB+ampi+arab flask. Ampicillin Preparation: # Create a stock solution of ampicillin of 100mg ampicillin to 1mL sterile water or transformation solution. Can store in refrigerator for later use. # Add ampicillin to LB+ampi and LB+ampi+arab: #* Concentration = 1 mL ampicillin stock solution / 1L LB agar. #* This gives a final concentration of 100uL ampi / 1mL agar. Arabinose Preparation: * Add arabinose to LB+a+a flask: ** Concentration = 2g arabinose / 1 L agar ** Arabinose requires at least 10 min to dissolve—be patient. Arabinose is shipped dry in a small vial. ** With a new sterile pipette, add 3 ml of transformation solution directly to the vial to rehydrate the sugar. ** Mix the vial; a vortex helps. (Transformation solution is being used here because it is a handy sterile solution. Sterile water would work just as well.) ** Discard pipette after use. Make Transformation solution Procedures # 7.3505 g CaCl2 dihydride # 1L DI H2O # Autoclave transformation solution (50mM CaCl2) Rehydrate HB101 E. coli bacteria Rehydrate and plate E. coli no more than 36 hours before making master plate For Bio-rad kit#1660555, rehydrate the E.coli per instructions and plate: # Using a sterile pipette, rehydrate the lyophilized E. coli HB101 by adding 250 μl of Transformation solution directly to the vial. Recap the vial and allow the cell suspension to stand at room temperature for 5 min. # Then shake to mix before streaking on LB or NA starter plates (Transformation solution is being used here because it is a handy sterile solution. Sterile water would work just as well.) # Store the rehydrated bacteria in the refrigerator until used (within 24 hours for best results, no longer than 3 days). Make E.coli LB starter plate - 2 days before * First make a master plate to make sure that there is enough bacteria, then make good streak plates for the students 24-36 hours before the class. Make E.coli LB student plates - 1 day before * Use starter plate to properly streak 1 LB of the pre-made / bench to obtain good isolated colonies * Incubate at 37 degrees for 24 hours * DO NOT REFRIGERATE for storage, store at room temp and use within 36 hours of incubation ending (NO more!). *** Cells need to be in the log phase of growth to transform correctly*** Aliquot solutions For each student bench aliquot: * 1 ml of transformation solution (50mM CaCl2) * 1 ml of LB nutrient broth into separate color-coded 2 ml microtubes provided in the kit. If the LB nutrient broth is aliquoted 1 day prior to the lab it should be refrigerated. Prepare pGLO plasmid * 250 μl of transformation solution into the vial of lyophilized pGLO plasmid DNA. * Invert the vial to mix plasmid solution well and ensure that the DNA is dissolved and is not sticking to the cap. If possible store the hydrated DNA in a refrigerator. Set up Lab Materials Day of Per bench * 1 plate competent HB101 E. coli * * 2 LB Plates* * 2 LB+ Ampi Plates* * 1 LB+Ampi+Arabinose plate* * 1 microtube of 1mL LB Broth* * 1 microtube 1 mL Transformation Solution (CaCl2) * 1 P1000 micropipette * P1000 Tips * 1 microtube holder * Ice for microtube holder * Sharpie * 2 microtubes * Loops * Bunsen burner Common workstation * 1 vial rehydrated pGLO plasmid (=10uL/group)* * 1 42°C water bath * Timers = 1/bench * 1 37°C incubator * 1 P20 micropipettes * 1 P20 micropipette tips * LAB 2 # Incubated plates # UV Light box (from chemistry) - optimal wavelength = 695nm? # UV Light SDS Sheet or Warning sheet